Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

Plant Methods

Klaus Harter¹  Frank Schleifenbaum³  Franziska Katharina Elisabeth Tiesler¹  Ying Zhou¹  Marko Vesić¹  Sébastien Peter³  Niklas Wallmeroth¹  Maik Böhmer²  Kenneth Wayne Berendzen¹ 

[1] Universität Tübingen, ZMBP, Plant Physiology, Auf der Morgenstelle 1, D-72076, Tübingen, Germany;University of California, San Diego, Division of Biological Sciences, Cell and Developmental Biology Section & Ctr for Mol. Genetics 0116, 9500 Gilman Drive #0116, La Jolla, CA, 92093-0116, USA;Universität Tübingen, ZMBP, Biophysical Chemistry, Auf der Morgenstelle 18, D-72076, Tübingen, Germany

关键词: CPK3;    Protein-protein interaction screen;    In vivo;    In planta;    BiFC;    FACS;   

Others  :  822026
DOI  :  10.1186/1746-4811-8-25

 received in 2012-04-17, accepted in 2012-05-26,  发布年份 2012

【 摘 要 】

Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first randomin planta screens to be successfully employed.

【 授权许可】

   
2012 Berendzen et al.; licensee BioMed Central Ltd.

【 图 表 】

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